Development of antibodies against the putative proteinase maturation protein A in relation to pneumococcal carriage and otitis media

The putative pneumococcal proteinase maturation protein A is a potential pneumococcal vaccine candidate. We examined serum antipneumococcal proteinase maturation protein A antibodies at 6, 12, 18 and 24 months of age, and showed that the age-related development of antipneumococcal proteinase maturation protein A antibodies is associated with pneumococcal contacts. A higher antipneumococcal proteinase maturation protein A antibody concentration at 18 months of age tends to predict for a lower risk of pneumococcal acute otitis media in the following 6 months (relative risk: 0.84, 95% confidence interval: 0.62-1.13).     

Modeling the structure of the StART domains of MLN64 and StAR proteins in complex with cholesterol

Steroidogenic acute regulatory protein-related lipid transfer (StART) domains are ubiquitously involved in intracellular lipid transport and metabolism and other cell-signaling events. In this work, we use a flexible docking algorithm, comparative modeling, and molecular dynamics (MD) simulations to generate plausible three-dimensional atomic models of the StART domains of human metastatic lymph node 64 (MLN64) and steroidogenic acute regulatory protein (StAR) proteins in complex with cholesterol. Our results show that cholesterol can adopt a similar conformation in the binding cavity in both cases and that the main contribution to the protein-ligand interaction energy derives from hydrophobic contacts. However, hydrogen-bonding and water-mediated interactions appear to be important in the fine-tuning of the binding affinity and the position of the ligand. To gain insights into the mechanism of binding, we carried out steered MD simulations in which cholesterol was gradually extracted from within the StAR model. These simulations indicate that a transient opening of loop Omega1 may be sufficient for uptake and release, and they also reveal a pathway of intermediate states involving residues known to be crucial for StAR activity. Based on these observations, we suggest specific mutagenesis targets for binding studies of cholesterol and its derivatives that could improve our understanding of the structural determinants for ligand binding by sterol carrier proteins.     

Immunomodulatory effects of thymopentin under acute and chronic inflammations in mice

The effects of thymopentin, a synthetic analog of the active center of the thymus hormone thymopoietin, on the immune status of mice with two different models of inflammation induced by injection of lipopolysaccharide (LPS) from Gram-negative bacteria were studied. Acute inflammation was induced by a single injection of LPS in a dose of 250 μg/100 g of body weight, and chronic inflammation (sepsis) was modeled by daily injection of LPS for 11 days with a gradual increase in the dose range from 25 to 250 μg/100 g of body weight. Under acute inflammation, a preliminary injection of thymopentin did not induce any additional stimulation of cytokine production increased by LPS. On the contrary, whereas the chronic introduction of LPS was characterized by a depressed production of several cytokines, thymopentin produced an immunostimulating effect. Thus an increase in the production of nitric oxide, interferon-μ, and Hsp70 was demonstrated. In addition, a more effective restoration of the number of thymus cells, as well as an increase in macrophage tumor necrosis factor-α production were observed after cessation of LPS + hormone injections. The results show that preliminary application of thymopentin promotes the regulation of immune cell activity under acute and chronic inflammation.     

Differential regulation of the STARD1 subfamily of START lipid trafficking proteins in human macrophages

The STARD1 subfamily of ‘START’ lipid trafficking proteins can reduce macrophage lipid content and inflammatory status (STARD1; StAR), and traffic cholesterol from endosomes (STARD3/MLN64). During macrophage differentiation, STARD1 mRNA and protein increase with sterol content, while the reverse is true for STARD3. Sterol depletion (methyl beta-cyclodextrin) enhances STARD3, and represses STARD1 expression. Agonists of Liver X receptors, peroxisome proliferator activated receptor-gamma and retinoic acid X receptors increase STARD1 expression, while hypocholesterolaemic agent, LY295427, reveals both STARD1 and STARD3 as putative SREBP-target genes. Pathophysiological ‘foam cell’ formation, induced by acetylated or oxidized LDL, significantly reduced both STARD1 and STARD3 gene expression. Differential regulation of STARD1 and D3 reflects their distinct roles in macrophage cholesterol metabolism, and may inform anti-atherogenic strategies.     

Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites

Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K_d = 0.15 μM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1″ phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.     

依达拉奉联合尤瑞克林治疗急性脑梗塞的疗效观察

目的:观察依达拉奉联合尤瑞克林治疗急性脑梗塞的疗效及安全性。方法:将84例急性脑梗塞患者随机分为2组,对照组予依达拉奉30 mg加入生理盐水100 mL静脉滴注,每日两次。观察组在上述基础上另予尤瑞克林0.15 PNA加入生理盐水250mL中静脉滴注,每天一次。两组疗程均为2周。观察两组患者的神经功能缺损评分、疗效与不良反应。结果:两组患者神经功能缺损评分均较治疗前明显改善(P<0.01),观察组评分明显低于对照组(P<0.01)。观察组临床总有效率达97.6%,明显高于对照组的57.1%(P<0.01)。两组不良反应发生率无统计学差异。结论:达拉奉联合尤瑞克林治疗急性脑梗塞临床疗效好,安全性高。     

Crystal Structures of the CERT START Domain with Inhibitors Provide Insights into the Mechanism of Ceramide Transfer

The cytosolic protein CERT transfers ceramide from the endoplasmic reticulum to the Golgi apparatus where ceramide is converted to SM. The C-terminal START (steroidogenic acute regulatory protein-related lipid transfer) domain of CERT binds one ceramide molecule in its central amphiphilic cavity. (1R,3R)-N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamide (HPA), a synthesized analogue of ceramide, inhibits ceramide transfer by CERT. Here we report crystal structures of the CERT START domain in complex with HPAs of varying acyl chain lengths. In these structures, one HPA molecule is buried in the amphiphilic cavity where the amide and hydroxyl groups of HPA form a hydrogen-bond network with specific amino acid residues. The Ω1 loop, which has been suggested to function as a gate of the cavity, adopts a different conformation when bound to HPA than when bound to ceramide. In the Ω1 loop region, Trp473 shows the largest difference between these two structures. This residue exists inside of the cavity in HPA-bound structures, while it is exposed to the outside of the protein in the apo-form and ceramide-bound complex structures. Surface plasmon resonance experiments confirmed that Trp473 is important for interaction with membranes. These results provide insights into not only the molecular mechanism of inhibition by HPAs but also possible mechanisms by which CERT interacts with ceramide.     

A novel recombinant snake venom metalloproteinase from Agkistrodon acutus protects against taurocholate-induced severe acute pancreatitis in rats

Severe acute pancreatitis (SAP) remains a challenging disease to manage, with high mortality, limited understanding of pathogenesis and lack of specific therapy. Recombinant fibrinogenase II (rFII) from Agkistrodon acutus venom has been found to degrade tumor necrosis factor-alpha (TNF-α) which is vital in mortality of SAP. Here we investigate the in vivo effects of rFII in rat SAP and confirm the degradation effect of rFII for TNF-α in vitro. The SAP model was prepared by retrograde infusion of 5% sodium taurocholate into the biliopancreatic duct in male Sprague–Dawley rats. Treatment with 1 mg/kg rFII could significantly increase survival rate of SAP rats (P = 0.006) as well as 8 mg/kg Infliximab treatment did. The pancreatic and pulmonary injury and the peritoneal and systemic inflammatory response were also attenuated by rFII as well as Infliximab. Furthermore, rFII inhibited TNF-α secretion by rat peritoneal macrophages in a time- and concentration-dependent manner but didn’t influence interleukin (IL) -1β secretion in vitro. The degradation potency of rFII for human TNF-α was greater than that for rat TNF-α. Our findings suggest that rFII could have protective effect on taurocholate-induced SAP in rats, mainly depending on direct degradation of TNF-α.